The invention relates to gene targeting for use, e.g., in the creation of transgenic, non-human mammals.
Gene targeting is a process whereby a specific gene, or a fragment of that gene, is altered. This alteration of the targeted gene may result in a change in the level of RNA or protein that is encoded by that gene, or the alteration may result in the targeted gene encoding a different RNA or protein than the untargeted gene. The targeted gene may be studied in the context of a cell, or, more preferably, in the context of a transgenic animal.
Transgenic animals are among the most useful research tools in the biological sciences. These animals have an heterologous (i.e., foreign) gene, or gene fragment, incorporated into their genome that is passed on to their offspring. Although there are several methods of producing transgenic animals, the most widely used is microinjection of DNA into single cell embryos. These embryos are then transferred into pseudopregnant recipient foster mothers. The offspring are then screened for the presence of the new gene, or gene fragment. Potential applications for transgenic animals include discovering the genetic basis of human and animal diseases, generating disease resistance in humans and animals, gene therapy, drug testing, and production of improved agricultural livestock.
In general, the invention features methods and uses for transposon-mediated gene targeting which greatly enhance the insertion and detection of desired genes in genomic exons by homologous recombination. The invention also features diagnostic methods for endocrine disorders, as well as methods and reagents for treating endocrine disorders.
In a first aspect, the invention provides a method for targeting heterologous DNA to integrate into an exon of a eukaryotic cell. The method includes, first, generating a pool of bacteria containing plasmids into which have been randomly integrated a transposon including heterologous DNA; second, isolating from the pool a bacterium which contains a plasmid into which the transposon is integrated into a copy of the exon on the plasmid by assessing PCR amplification products generated from the pool using primers specific for the exon; third, introducing the plasmid of the bacteria into the cell under conditions that promote homologous recombination; and, fourth, screening genomic DNA of the cell for integration of the heterologous DNA into the exon of the cell.
In one embodiment of the first aspect of the invention, the transposon bears at its extremities recognition sequences of a first rare-cutting restriction endonuclease that is absent in the exon. In another embodiment, the heterologous DNA, or portion thereof, encodes a selectable marker protein. The heterologous DNA, or portion thereof, may additionally encode a second protein, or polypeptide fragment thereof. In another embodiment, the marker protein is a prokaryotic selectable marker protein, which may be replaced by a eukaryotic selectable marker protein via the recognition sequences of the first rare-cutting restriction endonuclease. The prokaryotic selectable marker protein may be additionally replaced with DNA, or a portion thereof, encoding a second protein, or polypeptide fragment thereof.
In another embodiment of this aspect, the exon copy or portion thereof has at its borders destroyed recognition sequences of a second rare-cutting restriction endonuclease. In another embodiment, the genomic DNA is digested with the second rare-cutting restriction endonuclease. In yet another embodiment, the screening is carried out by Southern blot analysis of the genomic DNA with a detectable probe specific for the exon, or with a detectable probe external to the exon. The screening may also be carried out by PCR amplification of the genomic DNA with primers specific for the exon, or with primers external to, but surrounding the exon such that the PCR product includes the exon.
In a preferred embodiment of the first aspect of the invention, the insertion of the heterologous DNA into the exon results in a reduced level of expression of the protein encoded by the gene of the exon. The insertion of the heterologous DNA into the exon may also result in the expression of a truncated protein encoded by the gene of the exon, expression of a fusion protein encoded by the gene of the exon and the heterologous DNA, or portion thereof, or expression of a product, which may be a fusion protein, encoded by the heterologous DNA, or portion thereof.
In a second aspect, the invention provides a method for making a transgenic, non-human vertebrate animal containing heterologous DNA by first producing an embryonal cell of the non-human vertebrate animal with a targeted exon by first, generating a pool of bacteria containing plasmids into which have been randomly integrated a transposon including heterologous DNA; second, isolating from the pool a bacterium which contains a plasmid into which the transposon is integrated into a copy of the exon on the plasmid by assessing PCR amplification products generated from the pool using primers specific for the exon; third, introducing the plasmid of the bacteria into the embryonal cells under conditions that promote homologous recombination; and fourth, screening genomic DNA of the embryonal cells to identify an embryonal cell in which there has occurred integration of the heterologous DNA into the exon. The identified embryonal cell is then grown to generate the transgenic animal.
In one embodiment of the second aspect of the invention, the transposon bears at its extremities recognition sequences of a first rare-cutting restriction endonuclease that are absent in the exon. In another embodiment, the heterologous DNA, or portion thereof, encodes a selectable marker protein. The heterologous DNA, or portion thereof, additionally encodes a second protein, or polypeptide fragment thereof.
In another embodiment, the marker protein is a prokaryotic selectable marker protein which may be replaced by a eukaryotic selectable marker protein via the recognition sequences of the first rare-cutting restriction endonuclease. In another embodiment, the prokaryotic selectable marker protein is additionally replaced with DNA, or a portion thereof, encoding a second protein, or polypeptide fragment thereof.
In another embodiment, the exon copy or portion thereof has at its borders destroyed recognition sequences of a second rare cutting restriction endonuclease. Genomic DNA may be digested with the second rare-cutting restriction endonuclease. In another embodiment, the screening is carried out by Southern blot analysis of the genomic DNA with a detectable probe specific for the exon, or with a detectable probe external to the exon. The screening may also be carried out by PCR amplification of the genomic DNA with primers specific for the exon, or with primers external to, but surrounding the exon such that the PCR product includes the exon.
In a preferred embodiment of this aspect of the invention, the animal expresses a reduced level of the protein encoded by the gene of the exon. In another embodiment, the animal expresses a truncated protein encoded by the gene of the exon. In another embodiment, the animal expresses a fusion protein product encoded by the gene of the exon and the heterologous DNA, or portion thereof. In another embodiment, the animal expresses a product, which may be a fusion protein, encoded by the heterologous DNA, or portion thereof.
In a third aspect, the invention features a transposon that includes a selectable marker cassette including the selectable marker operably linked to a promoter, or hybrid thereof, capable of expressing the marker in both eukaryotic and prokaryotic cells. In a preferred embodiment of this aspect of the invention, the selectable marker is both a prokaryotic and eukaryotic selectable marker. In another embodiment of this aspect of the invention, the cassette is flanked by the recognition sequences of one or more rare-cutting restriction endonucleases. Most preferably, the transposon of this aspect of the invention is used to integrate a targeted gene, or exon thereof, on a plasmid.
In a fourth aspect, the invention features a eukaryotic cell containing an endogenous exon into which there is integrated a transposon including DNA encoding a selectable marker.
In a fifth aspect, the invention provides a method for making a transgenic non-human vertebrate animal by providing an embryonal cell of the non-human vertebrate animal that includes an endogenous exon into which there is integrated a transposon including DNA encoding a selectable marker, and then growing the cell to produce the transgenic animal.
In a sixth aspect, the invention features a transgenic non-human mammal having an abnormal genotype that gives rise to one or more symptoms of Cushing""s disease. In various embodiments, the mammal may have a disruption in an endogenous 7b2 gene, and the disruption may result in a reduced level of normal 7b2 protein. In another embodiment, the mammal is a mouse.
In a seventh aspect, the invention features a nucleic acid vector that includes nucleic acid capable of undergoing homologous recombination with an endogenous 7b2 gene, where the homologous recombination results in a disruption in the coding sequence of the endogenous 7b2 gene. In one embodiment, the disruption results in a reduced level of normal 7b2 protein.
In an eighth aspect, the invention features a eukaryotic cell containing an endogenous 7b2 gene into which there is integrated a transposon that includes DNA encoding a selectable marker.
In a ninth aspect, the invention features a method for diagnosing a mammal for an endocrine disorder that involves measuring 7b2 gene expression in a sample from the mammal, where abnormal expression is an indication that the mammal has an endocrine disorder or an increased likelihood of developing an endocrine disorder. Preferably, the mammal is a human.
In various embodiments of the ninth aspect of the invention, the abnormal expression is a reduced expression and the endocrine disorder is a hypercortisolism disorder (e.g., Cushing""s disease), or the abnormal expression is a reduced expression and the endocrine disorder is a hypoglycemic disorder. In other embodiments, the abnormal expression is an increased expression and the endocrine disorder is a hypocortisolism disorder (e.g., Addison""s disease), or the abnormal expression is an increased expression and the endocrine disorder is a hyperglycemic disorder (e.g., diabetes). In other embodiments, the expression is measured by assaying the amount of 7b2 polypeptide in the sample or by assaying the amount of 7b2 RNA in the sample.
In a tenth aspect, the invention features a method for diagnosing a mammal for an endocrine disorder or an increased likelihood of developing an endocrine disorder that involves isolating a sample of nucleic acid from the mammal and determining whether the nucleic acid includes a mutation in a 7b2 gene, where the presence of the mutation is an indication that the mammal has an endocrine disorder or has an increased likelihood of developing an endocrine disorder. Preferably, the mammal is a human.
In various embodiments of the tenth aspect of the invention, the mutation results in a reduced level of expression of the 7b2 gene and the endocrine disorder is a hypercortisolism disorder (e.g., Cushing""s disease), or the mutation results in a reduced level of expression of the 7b2 gene and the endocrine disorder is a hypoglycemic disorder. In other embodiments, the mutation results in an increased level of expression of the 7b2 gene and the endocrine disorder is a hypocortisolism disorder (e.g., Addison""s disease), or the mutation results in an increased level of expression of the 7b2 gene and the endocrine disorder is a hyperglycemic disorder (e.g., diabetes).
In an eleventh aspect, the invention features a method for determining whether a compound is potentially useful for treating or alleviating the symptoms of an endocrine disorder that includes the steps of: (a) providing a cell including a reporter gene operably linked to the promoter from a 7b2 gene; (b) contacting the cell with the compound; and (c) measuring the expression of the reporter gene, where a change in the level of the expression in response to the compound indicating that the compound is potentially useful for treating or alleviating the symptoms of an endocrine disorder.
In a twelfth aspect, the invention features a method for determining whether a compound is potentially useful for treating or alleviating the symptoms or an endocrine disorder that includes the steps of: (a) providing a cell expressing a 7b2 gene; (b) contacting the cell with the compound; and (c) monitoring the expression of the 7b2 gene, where a change in the level of the expression in response to the compound indicating that the compound is potentially useful for treating or alleviating the symptoms of an endocrine disorder.
In various embodiments of the eleventh and twelfth aspects of the invention, the change is a decrease and the endocrine disorder is a hypercortisolism disorder (e.g., Cushing""s disease), or the change is a decrease and the endocrine disorder is a hypoglycemic disorder. In other embodiments, the change is an increase and the endocrine disorder is a hypocortisolism disorder (e.g., Addison""s disease), or the change is an increase and the endocrine disorder is a hyperglycemic disorder (e.g., diabetes). In yet other embodiments, the 7b2 gene is from a mammal (e.g., a human or a mouse).
As used herein, by xe2x80x9cproteinxe2x80x9d or xe2x80x9cpolypeptidexe2x80x9d is meant any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).
By xe2x80x9cexonxe2x80x9d is meant a region of a gene which includes sequences which are used to encode the amino acid sequence of the gene product.
By xe2x80x9cknock-outxe2x80x9d is meant an alteration in the nucleic acid sequence that reduces the biological activity of the polypeptide normally encoded therefrom by at least 80% compared to the unaltered gene. The alteration may be an insertion, deletion, frameshift mutation, or missense mutation. Preferably, the alteration is an insertion or deletion, or is a frameshift mutation that creates a stop codon.
By xe2x80x9cplasmidxe2x80x9d is meant a circular strand of nucleic acid capable of autosomal replication in plasmid-carrying bacteria. The term includes nucleic acid which may be either DNA or RNA and may be single- or double-stranded. The plasmid of the definition may also include the sequences which correspond to a bacterial origin of replication.
By xe2x80x9crare-cutting restriction endonucleasexe2x80x9d is meant a restriction endonuclease whose recognition sequences are located at least 5,000 base pairs apart in the genomic DNA of a mammal. Such restriction endonucleases include, without limitation, SpeI, NotI, AscI, and PacI.
By xe2x80x9cdestroyed recognition sequencexe2x80x9d is meant the recognition sequence of a restriction endonuclease which has been destroyed such that the sequence is no longer recognized or cleaved by the restriction endonuclease. One means of generating a destroyed recognition sequence is to ligate cleaved ends of recognition sequences from two different restriction endonucleases. For example, a SpeI fragment may be ligated to an XbaI fragment creating ligated DNA having the sequence of 5xe2x80x2 ACTAGA 3xe2x80x2 (SEQ ID NO: 1), which is not recognized by either SpeI or XbaI.
By xe2x80x9coperably linkedxe2x80x9d is meant that a gene and a regulatory sequence are connected in such a way as to permit expression of the gene product under the control of the regulatory sequence.
By xe2x80x9cselectable markerxe2x80x9d is meant a gene product which may be selected for or against using chemical compounds, especially drugs. Selectable markers often are enzymes with an ability to metabolize the toxic drugs into non-lethal products. For example, the pac (puromycin acetyl transferase) gene product can metabolize puromycin, the dhfr gene product can metabolize trimethoprim (trnp) and the bla gene product can metabolize ampicillin (amp). Selectable markers may convert a benign drug into a toxin. For example, the HSV tk gene product can change its substrate, FIAU, into a lethal substance. A preferred selectable marker is one which may be utilized in both prokaryotic and eukaryotic cells. The neo gene, for example, metabolizes and neutralizes the toxic effects of the prokaryotic drug, kanamycin, as well as the eukaryotic drug, G418.
By xe2x80x9creporter genexe2x80x9d is meant any gene which encodes a product whose expression is detectable. A reporter gene product may have one of the following attributes, without restriction: fluorescence (e.g., green fluorescent protein), enzymatic activity (e.g., lacZ or luciferase), or an ability to be specifically bound by a second molecule (e.g., biotin or an antibody-recognizable epitope).
By xe2x80x9ctransgenicxe2x80x9d is meant any animal which includes a nucleic acid sequence which is inserted by artifice into a cell and becomes a part of the genome of the animal that develops from that cell. Such a transgene may be partly or entirely heterologous to the transgenic animal. Although transgenic mice represent a preferred embodiment of the invention, other transgenic mammals including, without limitation, transgenic rodents (for example, hamsters, guinea pigs, rabbits, and rats), and transgenic pigs, cattle, sheep, and goats are included in the definition.
By xe2x80x9ctransposonxe2x80x9d or xe2x80x9ctransposable elementxe2x80x9d is meant a linear strand of DNA capable of integrating into a second strand of DNA which may be linear or may be a circularized plasmid. Transposons often have insertion sequences, or remnants thereof, at their extremities, and are able to integrate into sites within the second strand of DNA selected at random, or nearly random. However, only one transposon may integrate into a second strand of DNA-following insertion of a transposon, the second strand of DNA becomes xe2x80x9ctransposition-incompetent.xe2x80x9d Preferred transposons have a short (e.g., less than ten) base pair repeat at either end of the linear DNA.
By xe2x80x9cendocrine disorderxe2x80x9d is meant a disorder affecting the endocrine system, resulting in an abnormally increased or reduced production of an endocrine hormone. Endocrine hormones include, without limitation, cortisol, corticosterone, insulin, and glucagon. Exemplary endocrine disorders include hypercortisolism (such as Cushing""s disease), hypocortisolism (such as Addison""s disease), hypoglycemia, and hyperglycemia (such as diabetes).